Allogeneic hematopoietic cell transplantation (AlloHCT) is used as corrective immunotherapy after ablative chemotherapies and has the potential to be applied for induction or restoration of immune tolerance. Unfortunately, graft-versus-host disease (GVHD), where donor T cells recognize and destroy alloantigen- expressing host tissues is a common AlloHCT-associated side effect that causes significant morbidity and mortality in cancer treatment. The risk of GVHD prevents the routine use of AlloHCT for tolerance induction. The OBJECTIVE of this application is to address these CLINICAL PROBLEMS using cutting-edge transgenic mouse models and identify targetable immune-mediated mechanisms that are able to consistently prevent or halt GVHD. To create space for donor stem cells and prevent their rapid rejection by host immune cells, AlloHCT protocols ablate recipient immune and stem cells with conditioning regimens involving irradiation or chemotherapy. These treatments damage mucosal barrier tissues and initiate the release of ?alarmins?, or typically sequestered self-derived immunomodulatory molecules that activate the immune system in poorly understood ways. Alarmins will also be released from recipient tissues damaged by allogeneic T cells during GVHD. We have used mice lacking the alarmin interleukin-33 (IL-33) or the IL-33 receptor, serum STimulation- 2 (ST2), to established that IL-33 signaling to donor T cells is required to drive the detrimental alloreactive T cell responses causing GVHD. ST2 deficient donor T cells are, however, able to mount a protective graft vs. leukemia (GVL) response. Our new data suggest that this may be because IL-33 plays a particularly important role in sustaining detrimental donor T helper Type 1 (Th1) response in the GVHD target tissues. Our ability to address the functions of IL-33 in GVHD and GVL has been complicated by the fact that IL-33 is expressed in the SLO, where alloimmune responses are initiated, and the mucosal barrier tissues, where GVHD manifests. Likewise, IL-33 has the potential to be active on T cells throughout GVHD development and the GVL response. We propose to use novel mice allowing us to separate out the temporal and spatial functions of IL-33 in rodent models of GVHD and GVL. This way we can test our HYPOTHESIS that IL-33 is critical to GVHD, but not GVL, because it is required to sustain the long-term effector functions and survival of donor Th1 responses in GVHD target tissues. We will use these innovative tools to test this hypothesis in two aims. In Aim 1 we will define the spatial and temporal importance of IL-33 signaling for the priming, differentiation, and effector function of alloreactive T cells mediating GVHD. In Aim 2 we will establish if disruption of IL-33 signaling to donor T cells in the peripheral tissues prevents GVHD, but preserves GVL. We expect to provide the first definitive evidence that IL-33 is a potent T cell stimulating alarmin whose dominant function is to sustain alloimmunity in GVHD target tissue, not the SLO. By using Tg mice allowing temporal disruption of T cell ST2 signaling, we also expect to establish when IL-33 or ST2 targeting therapeutics can be delivered to prevent or resolve GVHD, but not impede GVL.